1. Field of the Invention
This invention generally relates to methods for forming dyed microspheres and populations of dyed microspheres. Certain embodiments include activating a chemical structure coupled to a dye using heat or light to form a reaction intermediate in the presence of a microsphere such that the reaction intermediate covalently attaches to a polymer of the microsphere thereby coupling the dye to the polymer.
2. Description of the Related Art
The following descriptions and examples are not admitted to be prior art by virtue of their inclusion within this section.
Spectroscopic techniques are widely employed in the analysis of chemical and biological systems. Most often, these techniques involve the absorption or emission of electromagnetic radiation by the material of interest. One such application is in the field of microarrays, which is a technology exploited by a large number of disciplines including the combinatorial chemistry and biological assay industries. One company, Luminex Corporation of Austin, Tex., has developed a system in which biological assays are performed on the surface of variously colored fluorescent microspheres. One example of such a system is illustrated in U.S. Pat. No. 5,981,180 to Chandler et al., which is incorporated by reference as if fully set forth herein. These microspheres are interrogated in a fluid flow device by laser excitation and fluorescence detection of each individual microsphere as they pass at relatively high speed through a detection zone. These values may be easily exported to a database for further analysis.
In the above-mentioned system, fluorescent dyes are absorbed into the microspheres and/or bound to the surface of the microspheres. The dyes are generally chosen based on their ability to emit light at the wavelength of the selected detection window. Further, the detection windows are spaced apart by a certain number of wavelengths, and the dyes are typically designed to minimize the overlap of a dye's fluorescent signal within adjacent windows. In one example, by employing two detection windows and two dyes, each at 10 different concentrations, there would thus be 100 fluorescently distinguishable microsphere sets.
Conventional methods of dyeing microspheres by covalent and noncovalent reactions can be summarized as follows. For instance, dyed microspheres can be produced through dispersion polymerization by several methods. Dyes can be dissolved in the monomer prior to polymerization as described by Horak, D. et al. in J. Polym. Sci., Part A, Polym. Chem., 33, 2961-2968, 1995, which is incorporated by reference as if fully set forth herein. Microspheres can also be dyed after polymerization using an organic solvent to swell the particles and transport the dye into the particles. Examples of such dyeing methods are illustrated in U.S. Pat. No. 4,613,559 to Ober, U.S. Pat. No. 6,514,295 to Chandler et al., U.S. Pat. No. 6,528,165 to Chandler et al., U.S. Pat. No. 6,599,331 to Chandler et al., U.S. Pat. No. 6,632,526 to Chandler et al., and U.S. Pat. No. 6,649,414 to Chandler et al., which are incorporated by reference as if fully set forth herein.
Copolymerization of reactive dyes may be employed to obtain particles with chemically bound dyes. Examples of such methods are described by Winnik, F.M. et al. in Eur. Polym. J., 23,617-622, 1987, which is incorporated by reference as if fully set forth herein. Alternatively, the particles or microcarriers can be optically encoded by fluorescent dyes (or smaller dyed particles) that are covalently bound to their surfaces. Examples of such methods are described by Sebestyen F. et al. in J. Pept. Science, 4, 294-299, 1998, Egner, B.J. et al. in J. Chem. Soc. Chem. Commun., 8,735-736, 1977, Nanthakumar, A. et al. in Bioconj. Chem., 11, 282-292, 2000, and U.S. Pat. No. 6,268,222 to Chandler et al., all of which are incorporated by reference as if fully set forth herein.
C—H bond insertion by a carbene or nitrene moiety is a desirable pathway for establishing a stable covalent linkage between two organic molecules as described by Breslow, R., Scriven (Ed.), Azides and Nitrenes, Chapter 10, AP, New York, 1984, which is incorporated by reference as if fully set forth herein. The use of nitrene as the reactive intermediate has stimulated efforts to develop new reagents which undergo C—H insertion efficiently as described by Autrey, R. et al. in J. Am. Chem. Soc., 109, 5814, 1987, which is incorporated by reference as if fully set forth herein. Perfluorophenyl azides (PFPA) have been shown to exhibit improved C—H insertion efficiency over their non-fluorinated analogs. Examples of such azides are illustrated in U.S. Pat. No. 5,580,697 to Keana et al., U.S. Pat. No. 5,582,955 to Keana et al., U.S. Pat. No. 5,587,273 to Keana et al., and U.S. Pat. No. 6,022,597 to Keana et al. and U.S. Patent Publication No. US 2003/0017164 to Rajagopalan et al., all of which are incorporated by reference as if fully set forth herein.
The methods described above, however, do have a number of drawbacks. For example, dissolving a dye in monomers prior to polymerization usually results in reduced conversion and polydisperse particles (a mixture of different sized particles) due to radical chain termination by the dye during polymerization. In addition, the usefulness of copolymerization of reactive dyes is limited by the lack of dyes that are stable under radical polymerization conditions. In particular, reactive dyes can change color during polymerization as described by Yates, M. Z. et al. in Langmuir, 16(11), 4757-4760, 2000, which is incorporated by reference as if fully set forth herein.
Accordingly, in view of the present state of the art in covalent coupling of dyes to microspheres for generating solvent-fast or organotolerant beads, there remains a need for a suitable method for covalent dyeing of microspheres.
Various molecules containing multiple azide groups have been used to crosslink polymers. Heat and light have been used to convert azide moieties to reactive nitrene groups. For example, conversion of carbonyl azide using light is described in U.S. Pat. No. 3,278,305 to Urbain et al., which is incorporated by reference as if fully set forth herein. Conversion of sulfonyl azide at 175° C. is described in U.S. Pat. No. 3,507,829 to Bostick et al., which is incorporated by reference as if fully set forth herein. Conversion of aromatic azide using light is described in U.S. Pat. No. 3,887,379 to Clecak et al., which is incorporated by reference as if fully set forth herein. Conversion of PFPA using light is described by Cai et al. in Chem. Mater., 2,631, 1990, which is incorporated by reference as if fully set forth herein.
In the early 1970s, Kodak was assigned a series of patents for permanently dyeing hydrophobic polymers with various dyes (e.g., azo or quinone dyes) with appended sulfonyl azide moieties. The sulfonyl azide moiety was used to covalently attach dye molecules to a polymer substrate through the intermediate nitrene that is generated with heat or light. Unreacted dye molecules could be subsequently washed away. In particular, Great Britain Patent Nos. 1 344 991 to Holstead et al. and 1 344 992 to Holstead et al. describe the use of sulfonyl azide moieties attached directly to aromatic rings of various dyes and fluorescent brighteners. After heating or irradiation of dye-embedded-polymer (in the form of film or fiber), the dye forms a new bond with the polymer. Great Britain Patent No. 1 344 993 to Holstead et al. describes the use of similar reactive dyes as part of photographic materials. Great Britain Patent No. 1 406 996 to Shuttleworth describes the preparation of reactive dyes through the attachment of benzene sulfonic azide through a linker group to various dye molecules. Great Britain Patent No. 1 412 963 to Pullan describes the use of dyes with various reactive groups (including sulfonyl azide) in transfer printing onto fabrics or films. All of the Great Britain Patents described above are incorporated by reference as if fully set forth herein.
Other companies have patented the use of sulfonyl azide containing dyes for the fast dyeing of fibers and shape articles as described in U.S. Pat. No. 3,695,821 to Kuroki et al. and photographic dry copying as described in U.S. Pat. No. 3,674,480 to Kampfer et al., both of which are incorporated by reference as if fully set forth herein. Armstrong World Industries, Inc., Lancaster, Pennsylvania, received patents in the early 1980s relating to the use of sulfonyl azide containing compounds to impart permanent yellow and brown colors to articles as described in U.S. Pat. No. 4,322,211 to Hoyle et al., U.S. Pat. No. 4,415,334 to Hoyle et al., and U.S. Pat. No. 4,556,625 to Lenox et al., all of which are incorporated by reference as if fully set forth herein. In addition, several academic groups have explored the use of sulfonyl azide groups for the fast dyeing of polymer fibers as described by Ayyangar, N.R. et al., in Journal of the Society of Dyers and Colorists (JSDC), pp. 13-19 and 55-57, 1979, Griffiths, J. et al., JSDC, pp. 455-458, 1977, Griffiths, J. et al., JSDC, pp. 65-70, 1978, Milligan, B. et al., JSDC, pp. 352-356, 1978, and Modi et al., Dyes and Pigments, 23, pp. 25-32, 1993, which are incorporated by reference as if fully set forth herein.
The effect of substituents on the thermal decomposition of sulfonyl azides to nitrenes has been studied as described by Karamancheva, I. et al., Dyes and Pigments, 13, pp. 155-160, 1990, Ayyangar, N.R. et al., Indian J. of Chemistry, 30B, pp. 42-45, 1991, and Leffler, J.E. et al., J. Org. Chem., 28, pp. 902-906, 1963, which are incorporated by reference as if fully set forth herein. Studies into the basic mechanism of the sulfonyl azide reaction have also been reported as described by Abramovitch, R.A. et al., J. Org. Chem., 40, pp. 883-889, 1975, Abramovitch, R.A. et al., J. Org. Chem., 42, pp. 2920-2926, 1997, and Ulrich, J., J. Org. Chem., 40, pp. 802-804, 1975, which are incorporated by reference as if fully set forth herein.
Certain biological assays require the attachment of chemically synthesized biomolecules (e.g., oligonucleotides, peptides, or oligosaccharides). These biomolecules may be produced by automated synthesis on microsphere resins (non-dyed) in organic solvents. After synthesis, the biomolecules are cleaved from the support and then attached to dyed microspheres using aqueous methods. This process would be simpler and cheaper if the synthesis could be performed directly on dyed microspheres. However, currently used microspheres would exhibit a loss in dye if exposed to the organic solvents involved in the synthesis. Hence, “organotolerant” microspheres, or microspheres that would retain their fluorescent signatures even if exposed to organic solvents, are desired.
Methods have been developed to impart organotolerance to microspheres that would otherwise be organo-intolerant. For example, U.S. Pat. No. 6,528,165 to Chandler, which is incorporated by reference as if fully set forth herein, describes silicon and sugar based coatings that when formed on dyed microspheres could impart organotolerance to the dyed microspheres. In addition, other molecules (such as antigens or drug candidates) with relatively low solubility in water or aqueous solvents would be more easily coupled to microspheres if the coupling could be performed in alcoholic or dimethyl sulfoxide (DMSO) solvent.